oprF-GS-FLAG-LBT4-GS-LBT4-GS-LBT4

The capture part of our circuit. OprF can be expressed as an anchor on the cell membrane to make sure the LBT can combine with terbium (Tb3 +) on the surface of the bacteria.

Usage and biology

LBT（lanthanide binding tag） is a kind of protein which can bind with the lanthanide ions. With the help of OprF, our bacteria could express the LBT on its cell membrane and bind the lanthanide ions in the water. So once we put our bacteria into the water, the lanthanide ions in the water would be bound with the bacteria, and the concentration of the lanthanide ions in the water would decrease.

In this way, no matter how large the water volume is, we can put our bacteria in the water and let the bacteria bind the targeted ions in the polluted water. We can even link three specific LBTs for different water bodies. And as the pollution of lanthanide ions is serious, we think that the using of our production would be popular.

Characterization

This is one section for capture part.LBT4 can bind the lanthanide ions and it can be expressed at the cell membrane of E.coli with the help of OprF. We set the 'FLAG' between oprF and LBT for fluorescence detection. In our experiment, we let the LBT4 be expressed successfully which means that the lanthanide ions in the water would be bound and recycled effectively after we put the bacteria in the water.

Figure1:oprF-GS-FLAG-LBT-GS-LBT-GS-LBT

DNA Gel Electrophoretic

To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result.

Figure1-2:The result of electrophoretic shows that we have the right bands which are 1488bp.

Our target genes are 1488bp, and as the marker is DS5000, we could be sure that the bright bands in this picture are our target genes.

SDS-PAGE

After oprF-LBT4 is expressed successfully, we centrifuged the bacteria liquid and separated different proteins by SDS-PAGE.

Figure2:Protein Electrophoresis of OprF-3*LBT.OprF-3*LBT protein is about 31kDa.

Figure shows an obvious ~31kDa protein bands of OprF-3*LBT4 in test lane, which cannot be found in control lane. This result proves that the bacteria could express OprF-LBT4 successfully.

Fluorescence Detection

After the induction by IPTG, we use DYKDDDDK Tag (9A3) Mouse mAb (Binds to same epitope as Sigma's Anti-FLAG M2 Antibody) as the primary antibody and the Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) as the second antibody to figure out whether the cell surface display system works or not.

Figure3:Fluorescence detection of OprF-3*LBT at 488nm, OprF-LBT could excitated a bright fluorescence

Under fluorescence microscope, OprF-LBT4 can excitated a bright fluorescence.

Titration

After the induction by IPTG, we centrifuged the bacteria liquid and put 3ml bacteria liquid in each tube with 40ml TbCl3. After reacting for an hour in the shaker and centrifugation, we precipitate the supernatant with NaOH. We titrate the precipitate with HCl. Here is the result.

Figure4: The result of titration.

Obviously, the lanthanide ions have decreased after we use our bacteria which means that our design is feasible. The effect of binding lanthanide ions could be clearly found by our eyes.

Figure5: WT compares with 3*LBT

Improvement

To get the most efficient and effective capture effect, we designed to construct 12 different circuits with 12 different LBTs. Then we planned to test their binding force to get the best one. However we have not yet complete all the LBT construction in time. After competition of this year, HUST-China will get these 3 LBTs to be expressed and improve the efficiency of expression. If we make it, we will compare different LBTs and they will be different in the ability of binding the lanthanide ions so that we can choose suitable LBTs in different conditions.